In metazoans, the cohesin complex plays critical roles in many fundamental aspects of genome structure and maintenance. It ensures the accurate alignment and segregation of the products of DNA replication during cell division by tethering them together as they are made. It also promotes normal packaging of chromosomes into the interphase nucleus, both before and after DNA replication, which in turn ensures proper gene regulation. Finally, cohesin promotes DNA repair following both sporadic and programmed DNA double strand breaks. But how are these distinct activities of the cohesin complex controlled and integrated to ensure maintenance of sequence and structural integrity? This fundamental question is the basis for the experiments in this proposal. The association of cohesin with chromatin is dynamic: cohesin activity at a particular time and place reflects the collective outcome of both pro- and anti-cohesive activities. Changes in cohesin stability or chromatin binding occur in response to cell cycle progression, DNA damage signaling, and in response to certain developmental cues. In many cases, however, the signaling pathways that result in changes in cohesin binding are poorly understood. Modification of the Smc3 subunit of cohesin by members of the Eco1 family of acetyltransferases stabilizes the interaction of cohesin with chromatin. In vertebrates, there are two members of this family, Esco1 and Esco2, which we have shown play distinctly different roles in cohesin regulation. Esco2 is essential for establishing sister chromatid cohesion during DNA replication, while Esco1 modulates cohesin in its role in promoting normal chromosome architecture. With the experiments described here we will define how these two enzymes are regulated to generate very different outcomes using similar catalytic activity. In this proposal I describe a series of experiments using cell culture models, biochemistry, and functional analysis in Xenopus egg extracts to define how Esco1 and Esco2 are regulated to perform their unique functions. In Aim 1, we will define the molecular basis for the association of Esco2 with the replication apparatus. In Aims 2 and 3, we will define how chromatin structure is regulated by Esco1, and the impact of this regulation on DNA repair. In Aim 4 we will define how Esco1 impacts chromosome structure by analyzing nuclear assembly in G1. These experiments will elucidate in mechanistic detail how cohesin function is entrained by DNA replication and cell cycle progression to ensure proper sister chromatid tethering and a functional genomic landscape in interphase nuclei.